Bloodstream forms of African trypanosomes, which endocytose macromolecules exclusively
through their flagellar pockets, contain an acid phosphatase (AcP) activity in this organelle. In
the present thesis, AcP activity was demonstrated cytochemically in some intracellular vesicles
and on the surface of Trypanosoma congolense as well as in the flagellar pocket. Unlike other
trypanosomatids such as Leishmania spp. and Trichomonas spp., these trypanosomes, while
viable, did not release this enzyme into the surrounding medium.
In contrast to mammalian cells, the AcP in T. congolense was shown by cell fractionation to
be a non-lysosomal enzyme. The enzyme was mostly recovered in the microsomal and
cytosolic fractions which had 52.7% and 44.4% of the total activity, respectively. Further
separation of the microsomal fraction showed an association of AcP activity with vesicles
derived from the plasma membrane, Golgi apparatus and endoplasmic reticulum.
After ammonium sulfate precipitation and chromatography on a succession of columns
containing Sephacryl S-300, DEAE-cellulose and Sephadex G-75, two acid phosphatases
(AcPi and ACP2) were produced from the cytosolic fraction. A membrane-bound acid
phosphatase (ACP3) was isolated from the microsomal pellets extracted with Triton X-l 14 and
subjected to the above chromatographic procedures. The molecular mass of AcP 1 was higher
than 700 kDa. It had an isoelectric point of 4.7. AcP2 (pi 5.3) and AcP3 (pi 6.5) had
molecular masses of 33 and 320 kDa, respectively. AcPi and ACP3 were strongly inhibited by
vanadate while ACP2 was strongly inhibited by p-chloromercuribenzoate. None of the
enzymes was inhibited by tartrate but all were inhibited by NaF. The Km values for each of the
various substrates differed widely between the three AcPs indicating that the binding site of
each enzyme was distinct. The best of all the substrates tested was para-nitrophenyl
phosphate.
On non-denaturing gels the enzymes exhibited very high molecular masses but on denaturing
SDS-PAGE, two similar bands of activity, localised at 62 and 65 kDa, were observed in all
three AcP preparations. Thus the three isolated enzymes may be derived from the same base
62 and 65 kDa units. Differences between enzymes may be derived from differential
processing of the isoenzymes for different functions at different locations. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9783 |
| Date | 21 October 2013 |
| Creators | Tosomba, Omalokoho Médard. |
| Contributors | Coetzer, Theresa Helen Taillefer., Lonsdale-Eccles, John D. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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